Collagen is the most abundant protein in vertebrates, occurring in virtually every tissue, including skin, tendon, bone, blood vessel, cartilage, ligament, and teeth. Collagen serves as the fundamental structural protein for vertebrate tissues. Collagen abnormalities are associated with a wide variety of human diseases, including arthritis, rheumatism, brittle bones, atherosclerosis, cirrhosis, and eye cataracts. Collagen is also critically important in wound healing. Increased understanding of the structure of collagen, and of how its structure affects its stability, facilitates the development of new treatments for collagen-related diseases and improved wound healing treatments.
Collagen is a fibrous protein consisting of three polypeptide chains that fold into a triple helix, Jenkins and Raines Nat. Prod. Rep. 19: 49-59 (2002). Mammals produce at least 17 distinct polypeptide chains that combine to form at least 10 variants of collagen. In each of these variants, the polypeptide chains of collagen are composed of approximately 300 repeats of the tripeptide sequence Xaa-Yaa-Gly, where the first amino acid, Xaa, is often a proline (Pro) residue, the second amino acid, Yaa, is often a 4(R)-hydroxyproline (Hyp) residue, and the third amino acid is glycine. In connective tissue (such as bone, tendon, cartilage, ligament, skin, blood vessels, and teeth), individual collagen molecules are wound together in tight triple helices. These helices are organized into fibrils of great tensile strength, Jones and Miller, J. Mol. Biol., 218:209-219 (1991). Varying the arrangements and cross linking of the collagen fibrils enables vertebrates to support stress in one-dimension (tendons), two-dimensions (skin), or three-dimensions (cartilage).
In vertebrates, the collagen polypeptide is translated with the typical repeat motif being ProProGly. Subsequently, in vivo, the hydroxylation of Pro residues is performed enzymatically after collagen biosynthesis but before the chains begin to form a triple helix. Thus, hydroxylation could be important for both collagen folding and collagen stability (Raines Protein Sci. 15:1219-1225 (2006)). The hydroxyl group of Hyp residues has long been known to increase the thermal stability of triple-helical collagen, Berg and Prockop, Biochem. Biophys. Res. Comm., 52:115-120 (1973). For example, the melting temperature of a triple helix of (ProHypGly)10 chains is 58° C., while that of a triple helix of (ProProGly)10 chains is only 24° C., see: Sakakibara et. al., Biochem. Biophys. Acta, 303:198-202 (1973). In addition, the rate at which (ProHypGly)10 chains fold into a triple helix is substantially greater than the corresponding rate for (ProProGly)10 chains, see: Chopra and Ananthanarayanan, Proc. Natl. Acad. Sci. USA, 79:7180-7184 (1982).
In general, molecular modeling based on the structure of triple-helical collagen and conformational energy calculations suggest that hydrogen bonds cannot form between the hydroxyl group of Hyp residues and any main chain groups of any of the collagen molecules in the same triple helix, see: Okuyama et. al., J. Mol. Biol., 152:247-443 (1981). Also, several models include the hypothesis that hydroxyproline increases the stability of collagen. It is believed that the stability may be a result of a bridge of water molecules formed between the hydroxyl group and a main chain carbonyl group. For reviews of observations advancing this hypothesis, see: Suzuki et. al., Int. J. Biol. Macromol., 2:54-56 (1980), and Némethy, in Collagen, published by CRC press (1988), and the references cited therein.
However, there exists experimental evidence that is inconsistent with the “bridge of water molecule” model. For example, the triple helices of (ProProGly)10 and (ProHypGly)10 were found to be stable in 1,2-propanediol, and Hyp residues conferred added stability in these anhydrous conditions, Engel et. al., Biopolymers, 16:601-622 (1977), suggesting that water molecules do not play a part in the added stability of (ProHypGly)10. Notably, the frequency of Hyp could be too low to support such a water network in natural collagen. In the strands of human type-I collagen, an Xaa-HypGly sequence occurs in no more than four consecutive triads, and occurs in four consecutive triads only twice over >1000 residues. In addition, heat capacity measurements are inconsistent with collagen having more than one bound water per six Gly-X-Y units, Hoeve and Kakivaya, J. Phys. Chem., 80:754-749 (1976). There exists no prior definitive demonstration of the mechanism by which the hydroxyproline residues stabilize collagen triplexes. Therefore, the molecular basis for these observed effects is still not clear. However, recent structural studies have begun to shed light on the structure and stability of collagen's triple-helix, see Jenkins and Raines (2002).
An alternative to the “bridge of water molecule model” (Bella et. al., Science 266: 75-81 (1994)) is that of stereoelectronic effects. It is hypothesized that by using stereoelectronic effects electronegative oxygen preorganizes and places the main chain in the proper conformation for triple-helix formation. (Holmgren et. al., Nature 392:666-667 (1998)). The stereoelectronic effect explanation originates from the observation that replacing Hyp with (2S,4R)-4-fluoroproline (Flp) increases triple-helix stability; the fluoro group is strongly electron-withdrawing but cannot participate effectively in a putative hydrogen-bonded network. Similar results have been obtained with (2S,4R)-4-chloroproline. (Shoulders et. al, Biopolymers 89:443-454 (2008)). This explanation has been challenged by a host-guest study in which a single Hyp→Flp substitution was shown to destabilize a triple helix. (Periskov et. al., J. Am. Chem. Soc. 125:11500-11501 (2003)). A similar study has, however, reported a stabilization. (Malkar et. al., Biochemistry 41:6054-6064 (2002)). Therefore, it is still unclear whether Hyp stabilizes collagen by serving as a template for a water network or through stereoelectronic effects. A better understanding of how the structure of collagen contributes to its stability would facilitate the design of a collagen or collagen mimics having improved stability. A highly stable collagen substitute could advance the development of improved wound healing treatments.
In recent years, there have been exciting developments in wound healing, including the development of tissue engineering and tissue welding. For example, autologous epidermal transplantation for the treatment of burns was a significant advance in tissue engineering. Tissue engineering has also led to the development of several types of artificial skin, some of which employ human collagen as a substrate. However, a major problem associated with this treatment is the fragile nature of these grafts during and after surgery.
Tissue welding is a wound healing technique in which a laser is used to thermally denature the collagen in the skin at the periphery of a wound. The wound is reannealed by permitting the renaturation of the collagen. In the case of large wounds, a “filler” or solder is required to effect reannealing of the wound. Various materials, including human albumin, have been used as solders for this purpose. A good solder is resilient and is non-immunogenic and should preferably be capable of interaction with native collagen in adjacent sites.
Collagen is also used for a variety of other medical purposes. For example, collagen is used in sutures which can be naturally degraded by the human body and thus do not have to be removed following recovery. A sometimes limiting factor in the design of collagen sutures is the strength of the collagen fibers. A synthetic or a naturally occurring collagen that has been modified to exhibit greater strength would aid in the usage of such collagen sutures by relieving this limitation.
Researchers have been working on ways to increase the triple helix stability of collagen. For example, they have prepared a synthetic collagen mimic by replacing Pro in the Xaa position or Hyp in the Yaa position with 4(R)Fluoroproline (Flp) greatly increasing triple helix stability. (See U.S. Pat. No. 5,973,112 to Raines, which is incorporated herein by reference in its entirety; Holmgren et. al. (1998); and Holmgren et. al., Chem. Biol. 6: 63-70 (1999)). In contrast, it has also been shown that replacing Pro or Hyp in the Yaa position with the diastereomer 4(S)-fluoroproline (flp) greatly decreases stability, see: Bretscher et. al., J. Am. Chem. Soc. 123:777-778 (2001).
Other synthetic collagen mimics with increased stability compared to the triple helix of the native collagen have been prepared. Such collagen variants include 4(S)-fluoroproline (flp) in the Xaa position of the triple helical collagen tripeptide having the formula (Xaa Yaa Gly)n. This collagen mimic was found to have increased stability relative to the collagen-related triple helices (ProYaaGly)n, (hypYaaGly)n, and (HypYaaGly)n. (See U.S. Pat. No. 7,122,521 to Raines et. al., which is incorporated herein by reference in its entirety).
Also, Raines et. al. has disclosed additional synthetic collagen mimics with a tripeptide unit having the formula (Xaa-Yaa-Gly)n where one of the positions Xaa or Yaa is a bulky, non-electron withdrawing proline derivative. For example, such tripeptides can have the formula: (Xaa-Flp-Gly)n, where Xaa is (2S,4R)-4-alkylproline or a (2S,4R)-4-thioproline, where Flp is (2S,4R)-4-fluoroproline, and n is a positive integer. The alkylprolines suitable in the Xaa position include 4-methylproline, 4-ethylproline, 4-propylproline, 4-isopropylproline, or other longer alkylprolines. Alternatively, the Yaa position may also be (2S,4S)-4-alkyl proline or a (2S,4S)-4-thioproline. All of these synthetic collagens result in stronger more stable triple helixes than native collagen. (See U.S. Published App. No. 20070275897 to Raines et. al., also incorporated by reference herein in its entirety). Despite the recent advances in this field, the art continues to seek more desirable approaches to prepare a collagen having increased stability for use in biomaterials for the medical field, and in leather-related products prepared by the tanning industry.